Polysaccharide and protein-polysaccharide cross-linked hydrogels for soft tissue augmentation

ABSTRACT

Disclosed herein are cohesive soft tissue fillers, for example, dermal and subdermal fillers, based on hyaluronic acids and optionally including proteins. In one aspect, hyaluronic acid-based compositions described herein include zero-length cross-linked moieties and optionally at least one active agent. The present hyaluronic acid-based compositions have enhanced flow characteristics, hardness, and persistence compared to known hyaluronic acid-based compositions. Methods and processes of preparing such hyaluronic acid-based compositions are also provided.

RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.13/052,690, filed Mar. 21, 2011, which claims priority to U.S.Provisional Patent Application No. 61/316,283, filed on Mar. 22, 2010,the entire disclosure of each of these application being incorporatedherein by this reference.

BACKGROUND

Various injectable dermal filler products have been developed fortreating or correcting facial imperfections, for example, wrinkles andvolume loss due to the natural effects of aging. Injectable “dermalfillers” temporarily restore a smoother, more youthful appearance.

Ideally, dermal fillers are long-lasting, soft, smooth and naturalappearing when introduced into or beneath the skin. Further, theseproducts are preferably easy to introduce into a patient using a finegauge needle and a low extrusion force such that there will be minimaldiscomfort to the patient.

Collagen based soft tissue fillers were developed over 20 years ago, andfor some time, bovine collagen-based fillers were the only U.S. Food andDrug Administration (FDA)-approved dermal fillers. Because these earlydermal fillers were bovine-based, one of the main disadvantages to theiruse has been the potential for allergic reaction in patients.

In February 2003, human-derived collagen filler compositions receivedFDA approval. These collagens provide the advantage of a significantlyreduced risk of allergic reactions. Unfortunately, such human-derivedcollagen filler compositions tended to rapidly degrade shortly afterinjection.

In December 2003, the first hyaluronic acid (HA)-based dermal fillerdermal filler was approved by the FDA. This was rapidly followed by thedevelopment of many other HA-based dermal fillers.

HA, also known as hyaluronan, is a naturally occurring, water solublepolysaccharide, specifically a glycosaminoglycan, which is a majorcomponent of the extra-cellular matrix and is widely distributed inanimal tissues. HA has excellent biocompatibility and does not causeallergic reactions when implanted into a patient. In addition, HA hasthe ability to bind to large amounts of water, making it an excellentvolumizer of soft tissues.

The development of HA-based fillers which exhibit ideal in vivoproperties as well as ideal surgical usability has proven difficult. Forexample, HA-based fillers that exhibit desirable stability properties invivo, can be so highly viscous that injection through fine gauge needlesis difficult. Conversely, HA-based fillers that are relatively easilyinjected through fine gauge needles often have relatively inferiorstability properties in vivo.

Current hydrogel synthesis strategies perform cross-linking of HA underbasic conditions using small molecules to link the respective chainstogether. However, under these conditions the HA chains hydrolyze intoshorter fragments and small molecule linkers are introduced into thehydrogel.

It is an objective of the present invention to provide stable, elasticsoft tissue fillers with improved rheological properties.

SUMMARY

The present invention relates to soft tissue fillers, for example,dermal and subdermal filler compositions, hereinafter, sometimes,interchangeably referred to as “dermal fillers”, “soft tissue fillers”or “fillers”.

In one aspect of the invention, a composition is provided which is ahydrogel comprising a cross-linked biocompatible polymer havingzero-length cross-linked moieties. In some embodiments, the compositionfurther comprises at least one other active ingredient incorporated intothe cross-linked biocompatible polymer.

The hydrogel may be formed by reacting at least one cross-linkablebiocompatible polymer with at least one zero-length cross-linking agentat neutral pH. For example, the neutral pH can be between about 6.0 andabout 8.0, such as for example, between about 6.5 and about 7.5, such asfor example about 7.0.

In some embodiments, the cross-linkable biocompatible polymer ishyaluronic acid (HA) and the zero-length cross-linking agent is1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).

In some embodiments, the zero-length cross-linking agent is reacted withthe HA in the presence of N-hydroxysuccinimide (NHS), sulfo-NHS (orsulfonyl-NHS) or 4-dimethylaminopyridine (DMAP).

In some embodiments, the hydrogel further includes at least one secondcross-linked biocompatible polymer. The second cross-linkedbiocompatible polymer can be, for example, a protein such as elastin.

In some embodiments, the hydrogel can include an active agent selectedfrom the group consisting of enzyme inhibitors, anesthetic agents,medicinal neurotoxins, antioxidants, anti-infective agents,anti-inflammatory agents, ultraviolet (UV) light blocking agents, dyes,hormones, immunosuppressants, and combinations thereof.

The present invention further provides a method for making a hydrogelfor soft tissue augmentation. The method includes providing at least onecross-linkable biocompatible polymer; dissolving the at least onecross-linkable biocompatible polymer in a buffered solution wherein thebuffered solution maintains a pH from approximately 6.0 to approximately8.0; adding at least one zero-length cross-linking agent to the bufferedsolution to form a reaction mixture; and allowing the reaction mixtureto stand for a time appropriate to form a hydrogel suitable for softtissue augmentation.

Embodiments of the method of the invention can include one or more ofthe following features. The at least one cross-linkable biocompatiblepolymer can be HA. The buffered solution can be a phosphate bufferedsaline having a pH of between about 6.5 and about 7.5. The at least onezero-length cross-linking agent can be EDC.

In some embodiments, NHS is included in the adding step.

In some embodiments, the method includes a dialyzing step after theadding step.

In some embodiments, the method includes at least one secondcross-linkable biocompatible polymer. The second cross-linkedbiocompatible polymer can be, for example, a protein such as elastin.

In one aspect of the invention, the method includes adding an activeagent. The active agent may be an active agent selected from the groupconsisting of enzyme inhibitors, anesthetic agents, medicinalneurotoxins, antioxidants, anti-infective agents, anti-inflammatoryagents, ultraviolet light blocking agents, dyes, hormones,immunosuppressants, and combinations thereof.

In a specific embodiment of the invention, a method for making ahydrogel for soft tissue augmentation is provided which comprises thesteps of providing a 0.1 M phosphate buffered saline (PBS) solutionhaving a pH between approximately 6.5 and approximately 7.5; dissolvingfrom about 20 to about 80 mg/mL HA from about 2 to about 20 weightpercent soluble elastin in the PBS to form a polymer mixture; addingabout 5 to about 30 mol percent EDC and NHS to the polymer mixture toform a reaction mixture; allowing the reaction mixture to react forabout 12 to about 48 hours at about 22° to about 60° C. to form a gel;dialyzing the gel against PBS to form a purified gel; and sizing thepurified gel to form a hydrogel for soft tissue augmentation. In someembodiments, the method includes adding an active agent selected fromthe group consisting of enzyme inhibitors, anesthetic agents, medicinalneurotoxins, antioxidants, anti-infective agents, anti-inflammatoryagents, ultraviolet light blocking agents, dyes, hormones,immunosuppressants, and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically illustrates the elasticity and viscosity of EDC/NHScross-linked HA and HA-elastin in accordance with an embodiment of theinvention.

FIG. 2 graphically illustrates the resistance of HA and HA-elastin toenzyme degradation.

FIG. 3 graphically illustrates the extrusion force of EDC/NHScross-linked HA and HA-elastin in accordance with an embodiment of theinvention.

DETAILED DESCRIPTION

Where the definition of terms as used in the specification departs fromthe commonly used meaning of the term, applicant intends to utilize thedefinitions provided herein, unless specifically indicated.

The present description generally relates to soft tissue fillers, forexample, dermal and subdermal fillers, based on cross-linkedbiocompatible polymers. In one aspect, the compositions described hereininclude hydrogels comprising at least one cross-linked biocompatiblepolymer having zero-length cross-linked moieties and optionally at leastone other active ingredient incorporated into the cross-linkedbiocompatible polymer. The present compositions, which include HA-basedhydrogels, have enhanced rheology (i.e., flow characteristics),elasticity, and persistence relative to known HA-based hydrogels.Methods or processes of preparing such compositions are also provided,as well as products made by such methods or processes.

A surprising advantage of the compositions and methods of the presentdescription is that the molecular weight of the polymer chains, such asHA, remains high and the resultant hydrogels have improved rheologicalproperties while at the same time have low extrusion forces. Hydrogelswith fine-tuned hardness and elasticity are beneficial for thedevelopment of biomaterials suitable for soft tissue augmentation.

In various embodiments, hydrogels of the present description include HAas a biocompatible polymer and are therefore HA-based. HA-based as usedherein refers to compositions including cross-linked HA and compositionsincluding cross-linked HA plus one or more other cross-linked polymers.In addition, HA can refer to hyaluronic acid and any of its hyaluronatesalts, including, but is not limited to, sodium hyaluronate (NaHA),potassium hyaluronate, magnesium hyaluronate, calcium hyaluronate, andcombinations thereof. The use of more than one biocompatible polymer isspecifically not excluded from the present description. Hydrogels of thepresent description can include more than one biocompatible polymer,such as, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more biocompatiblepolymers. Suitable biocompatible polymers include polysaccharides (e.g.,HA, chitosan, chondroitin sulfate, alginate, carboxymethylcellulose),poly(ethyleneglycol), poly(lactic acid), poly(hydroxyethylmethacrylate),poly(methylmethacrylate), proteins (e.g., elastin and collagen).

Generally, the HA concentration in the compositions described herein ispreferably at least 10 mg/mL and up to about 100 mg/mL. For example, theconcentration of HA in some of the compositions is in a range betweenabout 15 mg/mL and about 80 mg/mL, or about 15 mg/mL to about 30 mg/mL.In some embodiments, the concentration of HA is about 26 mg/mL. In someembodiments, hydrogels are formed by reacting at least onecross-linkable biocompatible polymer, such as HA and/or protein, with atleast one zero-length cross-linking agent. Generally, zero-lengthcross-linking agents couple polymers without adding any additionalspacer arm atoms and therefore zero-length cross-linking agents are notincorporated into the cross-linked polymer matrix. Suitable zero-lengthcross-linking agents include carbodiimides, such as, for example,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Non-water solublecarbodiimides include dicyclohexylcarbodiimide (DCC) anddiisopropylcarbodiimide (DIC), which may also be suitable.

Carbodiimide-mediated coupling between carboxylates and alcohol or aminefunctional groups proceeds readily at ambient temperature, neutral pHand under aqueous conditions. Neutral pH can be, for example, betweenabout 6.0 and about 8.0, such as between about 6.5 and about 7.5, suchas about 7.0. Typically in water, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) can be used to mediate esterificationbetween carboxylates and alcohols or amidation between carboxylates andamines. Thus, cross-linked HA is formed by exploiting reactive groupspresent on HA (e.g, carboxylate and alcohol). In addition, by takingadvantage of the high reactivity of amine groups on proteins, amidationbetween lysine side-chains of proteins with carboxylate groups of HA isachieved to form HA-protein cross-linked hydrogels. Cross-linking agentsand unreacted polymers can be removed by dialysis.

In some embodiments, EDC is used in conjunction withN-hydroxysuccinimide (NHS) or sulfonyl-NHS (sulfo-NHS), collectivelyreferred to as “NHS” herein. NHS stabilizes reactive intermediatesformed by EDC; thus, the addition of NHS can increase the couplingefficiency of EDC. Alternatively, 4-dimethylaminopyridine (DMAP) can beused to catalyze the coupling reaction.

Without being limited thereto, the HA-based compositions in accordancewith the present description include cross-linked HA-based compositionsand at least partially cross-linked HA-based compositions.Uncross-linked HA as used herein refers to both truly uncross-linked(e.g., “free”) HA chains as well as lightly cross-linked chains andfragments thereof that are generally in soluble liquid form.

The soft tissue fillers of the present description can include an activeagent selected from the group consisting of enzyme inhibitors,anesthetic agents, medicinal neurotoxins (e.g., botulinum toxin andclostridium toxin), antioxidants, anti-infective agents (e.g.,antibiotics), anti-inflammatory agents, ultraviolet (UV) light blockingagents, dyes, hormones, immunosuppressants, and combinations thereof.

In addition, the soft tissue fillers of the present invention caninclude one or more anesthetic agents in an amount effective to mitigatepain experienced upon injection of the composition. The local anestheticcan be selected from the group of ambucaine, amolanone, amylocaine,benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine,butacaine, butamben, butanilicaine, butethamine, butoxycaine,carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine,dibucaine, dimethisoquin, dimethocaine, diperodon, dycyclomine,ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine,euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine,isobutyl p-aminobenzoate, leucinocaine mesylate, levoxadrol, lidocaine,mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine,naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine,phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine,prilocaine, procaine, propanocaine, proparacaine, propipocaine,propoxycaine, pseudococaine, pyrrocaine, ropivacaine, salicyl alcohol,tetracaine, tolycaine, trimecaine, zolamine, and salts thereof. In oneembodiment, the anesthetic agent is lidocaine, such as in the form oflidocaine HCl. The compositions described herein may have a lidocaine orother anesthetic in a concentration of between about 0.1% and about 5%by weight of the composition, for example, about 0.2% to about 1.0% byweight of the composition. In one embodiment, the composition has alidocaine concentration of about 0.3% by weight (w/w %) of thecomposition. The concentration of lidocaine in the compositionsdescribed herein can be therapeutically effective meaning theconcentration is adequate to provide a therapeutic benefit.

The present description also provides methods for making hydrogels. Themethods can include providing at least one cross-linkable biocompatiblepolymer, such as HA. The initial step of providing raw HA material canbe in the form of dry HA fibers or powder. The raw HA material may beHA, its salts and/or mixtures thereof. The HA material can comprise, forexample, NaHA fibers or powder of bacterial-sourced NaHA. In someaspects of the present description, the HA material may be animalderived. The HA material may be a combination of raw materials includingHA and at least one other polysaccharide, for example, glycosaminoglycan(GAG).

In some embodiments, the HA material in the compositions nearly entirelycomprises or consists of high molecular weight HA. That is, nearly 100%of the HA material in the present compositions may be high molecularweight HA. In other embodiments, the HA material in the compositionscomprises a combination of high molecular weight HA and low molecularweight HA.

The HA material of the compositions may comprise between about 5% toabout 95% high molecular weight HA with the balance of the HA materialincluding low molecular weight HA. In a typical composition according tothe present description, the ratio of high molecular weight to lowmolecular weight HA is at least about, and preferably greater than 2(w/w≧2) with the high molecular weight HA having a molecular weight ofabove about 1.0 MDa.

It will be appreciated by those of ordinary skill in the art that theselection of high and low molecular weight HA material and theirrelative percentages or ratios is dependent upon the desiredcharacteristics, for example, extrusion force, elastic modulus, viscousmodulus and persistence of the final HA-based product. For additionalinformation that may be helpful in understanding this and other aspectsof the present description, see U.S. Patent Application Publication No.2006/0194758, the entire description of which is incorporated herein bythis reference.

HA-based gels can be prepared according to the present description byfirst cleaning and purifying dry or raw HA material having a desiredhigh/low molecular weight ratio. These steps generally involve hydratingthe dry HA fibers or powder in the desired high/low molecular weightratio, for example, using pure water, and filtering the material toremove large foreign matters and/or other impurities. The filtered,hydrated material is then dried and purified. The high and low molecularweight HA may be cleaned and purified separately, or may be mixedtogether, for example, in the desired ratio, just prior tocross-linking.

Methods of making hydrogels in accordance with the present descriptioncan include the step of dissolving the cross-linkable biocompatiblepolymer in a buffered solution. In some embodiments, the bufferedsolution is maintained at a pH between about 6.0 and about 8.0, such asbetween about 6.5 and about 7.5, such as at about 7.0. In a preferredembodiment, the biocompatible polymer is HA and the buffer is phosphatebuffered saline (PBS). In some embodiments, a second biocompatiblepolymer is added. When more than one biocompatible polymer is used, thepolymers can be added in ratios which yield suitable flowcharacteristics, elasticity, viscosity, and persistence. In a preferredembodiment, the second biocompatible polymer is a protein such aselastin. In a particularly preferred embodiment, about 20 to about 80mg/mL HA is dissolved in the buffer solution, and about 2 to about 20weight percent of soluble elastin is dissolved in the buffer solution.

The methods of making hydrogels in accordance with the presentdescription can include the step of adding at least one zero-lengthcross-linking agent to the buffered solution. The use of more than onecross-linking agent or a different cross-linking agent is not excludedfrom the scope of the present description. In a preferred embodiment,the zero-length cross-linking agent is EDC. NHS can be added to thereaction mixture before, or together with EDC to increase cross-linkingefficiency. EDC and NHS can be added in any ratio, such as 10:1, 5:1,2:1, 1:1, 1:2, 1:5, and 1:10. In a preferred embodiment, EDC and NHS areadded in one portion (i.e., 1:1 ratio). EDC and NHS can be prepared atany suitable concentration, such as between about 1 to about 50 molpercent, and more preferably between about 5 to about 30 mol percent.

The reaction mixture is allowed to stand for a time appropriate to forma hydrogel suitable for soft tissue implantation. In some embodiments,the time is from between about 12 hours to about 72 hours, such asbetween about 12 and about 48 hours, such as about 24 hours. In someembodiments, the reaction mixture is maintained at an appropriatetemperature, such as between about 22 to about 60 degrees Celsius.

The step of cross-linking may be carried out using any means known tothose of ordinary skill in the art. Those skilled in the art appreciatehow to optimize conditions of cross-linking according to the nature ofthe biocompatible polymers, and how to carry out cross-linking to anoptimized degree. A degree of cross-linking is preferably sufficient forthe final hydrogel composition obtained from the present methods toremain implanted at the injection site without excessive diffusion awayfrom the injection site. In some embodiments, the degree ofcross-linking is at least about 2% to about 20%, and more preferably isabout 4% to about 12%, wherein the degree of cross-linking is defined asthe percent weight ratio of the cross-linking agent to HA-monomericunits in the composition. The degree of cross-linking can be less thanabout 6% or more preferably less than about 5%.

The method of making a hydrogel can include the step of dialyzing thehydrogel after the hydrogel is formed to remove cross-linking agents,unreacted starting materials, and by-products such as N-acylurea. Insome embodiments, the dialysis is performed using a buffer solution. Ina preferred embodiment, the buffer solution is PBS. The purifiedhydrogel can then be sized using any suitable method, such as by passingthrough a stainless steel screen having the desired mesh size.

The cross-linked, HA-based gels can comprise a cross-linked HA componentcapable of absorbing at least about one time its weight in water. Whenneutralized and swollen, the cross-linked HA component and waterabsorbed by the cross-linked HA component is in a weight ratio of about1:1.

The compositions described herein display an elastic modulus and viscousmodulus which is dependent on the specific biocompatible polymers usedand the presence and/or absence of at least one active agent. In someembodiments, the elastic modulus of the HA-based compositions can be atleast about 50 Pa, but is more preferably between about 150 Pa and about1500 Pa, such as between about 500 Pa to about 1200 Pa. In someembodiments, the viscous modulus can be between about 50 Pa to about 500Pa, such as between about 50 Pa to about 200 Pa. In one exemplaryembodiment, the viscous modulus is about 160 Pa.

In some embodiments, the method includes the step of adding an activeingredient to the hydrogel. As discussed in more detail above, theactive ingredient can be selected from the group consisting of enzymeinhibitors, anesthetic agents, medicinal neurotoxins, antioxidants,anti-infective agents, anti-inflammatory agents, ultraviolet lightblocking agents, dyes, hormones, immunosuppressants, and combinationsthereof. The use of more than one active agent is specifically notexcluded from the present description. In some embodiments, one or moreactive agents are added to the purified hydrogel. In other embodiments,one or more active agents are added to the reaction buffer and arecross-linked with the biocompatible polymer and/or the active agents areentrapped or encased by the cross-linked biocompatible polymer.

Syringes useful for administering the hydrogels of the presentdescription include any syringe known in the art capable of deliveringviscous dermal filler compositions. The syringes generally have aninternal volume of about 0.4 mL to about 3 mL, more preferably betweenabout 0.5 mL and about 1.5 mL or between about 0.8 mL and about 2.5 mL.This internal volume is associated with an internal diameter of thesyringe which affects the extrusion force needed to inject highviscosity dermal filler compositions. The internal diameters aregenerally about 4 mm to about 9 mm, more preferably from about 4.5 mm toabout 6.5 mm or from about 4.5 mm to about 8.8 mm. Further, theextrusion force needed to deliver the HA-based compositions from thesyringe is dependent on the needle gauge. The gauges of needles usedgenerally include gauges between about 18 G and about 40 G, morepreferably about 25 G to about 33 G or from about 16 G to about 25 G. Aperson of ordinary skill in the art can determine the correct syringedimensions and needle gauge required to arrive at a particular extrusionforce requirement.

The extrusion forces displayed by the HA-based compositions describedherein using the needle dimensions described above are applied usinginjection speeds that are comfortable to a patient. Comfortable to apatient is used to define a rate of injection that does not injure orcause excess pain to a patient upon injection to the soft tissue. Oneskilled in the art will appreciate that comfortable as used hereinincludes not only patient comfort, but also comfort and ability of thephysician or medical technician injecting the HA compositions. Althoughcertain extrusion forces may be achievable with the HA compositions ofthe present description, one skilled in the art understands that highextrusion forces can lead to lack of control during injection and thatsuch lack of control may result in additional pain to the patient.Extrusion forces of the present HA compositions can be from about 8 N toabout 40 N, or more preferably from about 10 N to about 30 N, or about15 N to about 20 N.

Sterilization, as used herein comprises any method known in the art toeffectively kill or eliminate transmissible agents, preferably withoutsubstantially altering or degrading the HA-based compositions and anyactive agents.

One preferable method of sterilization of the filled syringes is byautoclave. Autoclaving can be accomplished by applying a mixture ofheat, pressure and moisture to a sample in need of sterilization. Manydifferent sterilization temperatures, pressures and cycle times can beused for this step. For example, the filled syringes may be sterilizedat a temperature of at least about 120° C. to about 130° C. or greater.Moisture may or may not be utilized. The pressure applied is in someembodiments depending on the temperature used in the sterilizationprocess. The sterilization cycle may be at least about 1 minute to about20 minutes or more.

Another method of sterilization incorporates the use of a gaseousspecies which is known to kill or eliminate transmissible agents.Preferably, ethylene oxide is used as the sterilization gas and is knownin the art to be useful in sterilizing medical devices and products.

A further method of sterilization incorporates the use of an irradiationsource which is known in the art to kill or eliminate transmissibleagents. A beam of irradiation is targeted at the syringe containing theHA solution, and the wavelength of energy kills or eliminates theunwanted transmissible agents. Preferable energy useful include, but isnot limited to ultraviolet light, gamma irradiation, visible light,microwaves, or any other wavelength or band of wavelengths which killsor eliminates the unwanted transmissible agents, preferably withoutsubstantially altering of degrading the HA-based composition or anyactive agent.

EXAMPLE 1 EDC-Mediated Cross-Linked Polysaccharide

Polysaccharide hydrogels were generated by cross-linking HA using EDCand NHS (or sulfo-NHS, collectively “NHS”).

Carbodiimide-mediated coupling of HA is performed in 0.1 M PBS atneutral pH (6.5-7.5). HA is dissolved in buffer (20-80 mg/mL). 5-30 mol% EDC and NHS are then added in one portion. Next, the polymer isallowed to cross-link over 12-72 hours at 22-60° C. The resulting gelmay be diluted and is then dialyzed extensively at ambient temperatureagainst PBS to remove N-acylurea byproducts and any unused startingmaterials. Sizing of the purified gel is then performed through astainless steel screen.

EXAMPLE 2 EDC-Mediated Cross-Linked Polysaccharide-Protein

Polysaccharide-protein hydrogels were generated by cross-linking HA andelastin using EDC and NHS.

Carbodiimide-mediated coupling of HA and proteins (e.g. elastin) isperformed in 0.1 M PBS at neutral pH (6.5-7.5). HA (20-80 mg/mL) andsoluble elastin (2-20 wt %) are dissolved in PBS. 5-30 mol % EDC and NHSare added in one portion. Next, the polysaccharide and protein areallowed to cross-link at ambient temperature over 12-48 hours at 22-60°C. The resultant gel is then dialyzed extensively at ambient temperatureagainst PBS to remove the N-acylurea byproducts and starting materials.Sizing of the purified gel is then performed through a stainless steelscreen.

EXAMPLE 3 Rheology of Cross-Linked HA and HA-Elastin

The flow characteristics of the hydrogels prepared according to Examples1 and 2 were evaluated in order to assess their elasticity andviscosity.

A strain sweep analysis provides information about a gel's elasticmodulus (G′) and viscous modulus (G″). A large value of G′ compared toG″ indicates a very elastic gel. FIG. 1 depicts typical strain sweepdata for a hydrogel comprised of EDC/NHS cross-linked HA and HA-elastin.The G′ for the two gels is 1100 Pa and 505 Pa, respectively where asthat of BDDE crosslinked HA is 150 Pa. The G′/G″ of the EDC/NHScross-linked HA is 6.9 and that of HA-elastin is 3.7, respectively. Incomparison, the G′/G″ of BDDE-crosslinked HA is approximately 2.5. Takentogether, these data suggest that the resulting gels from EDC/NHSmediated cross-linking have higher elasticity and hardness compared toBDDE-crosslinked hydrogel. Accordingly, EDC/NHS cross-linked HA andHA-proteins have improved properties suitable for soft tissueaugmentation, such as, for example, dermal filling.

EXAMPLE 4 Enzyme Degradation of Cross-Linked HA and HA-Elastin

Hydrogels prepared according to Examples 1 and 2 were subjected toenzyme degradation in vitro to assess resistance to enzymaticdegradation.

It is generally understood that improved resistance to enzymedegradation correlates to improved in vivo persistence. A superior gelresists degradation and therefore has a smaller difference of free HAbefore and after enzyme degradation. First, the percentage ofuncross-linked (i.e., “free” HA) before and after enzymatic degradationwith bovine testicular hyaluronidase (HAse) is measured by sizeexclusion chromatography (SEC) on an Agilent (Santa Clara, Calif.) HPLCsystem equipped with multi-angle laser light scattering (MALS) andrefractive index detectors. FIG. 2 depicts enzymatic degradation ofEDC/NHS cross-linked HA and HA-elastin, and BDDE-crosslinked HAhydrogel. As shown in FIG. 2, the increase in soluble HA is 37 and 4%for the EDC/NHS cross-linked HA and HA-elastin, respectively. Theincrease in soluble HA for BDDE crosslinked HA is 41%. Thus, HA-elastinresists enzymatic degradation more than the EDC/NHS cross-linked HA, andboth HA and HA-elastin resist degradation more than BDDE crosslinked HA.As a result, EDC/NHS cross-linked HA and HA-proteins are expected tohave improved in vivo persistence.

EXAMPLE 5 Extrusion of Cross-Linked HA and HA-Elastin

The extrusion force of hydrogels prepared according to Examples 1 and 2were determined to evaluate the feasibility of administering thehydrogels through a needle.

Extrusion force analysis was performed on an INSTRON® (Norwood, Mass.)instrument using a 0.8 mL syringe equipped with a 30 gauge (G) needle.The hydrogel was extruded at a constant rate of 50 mm/min. Shown in FIG.3 is a graph of compressive extension plotted against compressive forcefor EDC/NHS cross-linked HA and HA-elastin as compared to BDDEcrosslinked HA. These data show that the maximum average compressiveforce of the EDC/NHS cross-linked HA and HA elastin are 37 N and 13 N,respectively, while that of BDDE crosslinked HA is 38 N. Accordingly,EDC/NHS cross-linked HA and HA-elastin can be administered via injectionthrough a 30 G needle.

EXAMPLE 6 Total Uncross-Linked HA

Total uncross-linked HA (i.e., free HA) was determined in the hydrogelsprepared according to Examples 1 and 2.

The percentage of uncross-linked HA is an important parameter inevaluating relative cross-linking efficiency, as well as to predict thein vivo persistence of a hydrogel. It is well documented thatuncross-linked HA is rapidly degraded in vivo; therefore, hydrogels witha larger percentage of uncross-linked HA are likely to have less in vivopersistence. Total uncross-linked HA is measured by diluting the gel 20×in PBS, and then allowing the gel to swell over 1 week with constantagitation. The solution is then filtered through a 0.22 μm filter toremove particulate and gel matter, and is then analyzed by SEC-MALS tomeasure the percentage of recovered HA. In this specific example, theEDC/NHS cross-linked HA and HA-elastin contain approximately 10% and 41%total uncross-linked HA, respectively. These data indicate that bothEDC/NHS cross-linked HA and HA-elastin are expected to have suitable invivo persistence.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe specification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Although the invention has been described and illustrated with a certaindegree of particularity, it is to be understood that the presentdisclosure has been made only by way of example, and that numerouschanges in the combination and arrangement of parts can be resorted toby those skilled in the art without departing from the scope of theinvention, as hereinafter claimed

What is claimed is:
 1. A method for making a hydrogel for soft tissueaugmentation, the method comprising: providing at least onecross-linkable biocompatible polymer; dissolving said at least onecross-linkable biocompatible polymer in a buffered solution, whereinsaid buffered solution maintains a pH from approximately 6.0 toapproximately 8.0; adding at least one zero-length cross-linking agentto said buffered solution to form a reaction mixture; and allowing saidreaction mixture to stand for a time appropriate to form the hydrogelsuitable for soft tissue augmentation, wherein said hydrogel comprises across-linked biocompatible polymer having zero-length cross-linkedmoieties; wherein at least one said cross-linkable biocompatible polymeris hyaluronic acid; and wherein said zero-length cross-linking agent is1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).
 2. The methodaccording to claim 1 further wherein said buffered solution is phosphatebuffered saline having a pH of between 6.5 and 7.5.
 3. The methodaccording to claim 1 further including N-hydroxysuccinimide (NHS) insaid adding step.
 4. The method according to claim 1 further comprisinga dialyzing step after said allowing step.
 5. The method according toclaim 1 further comprising at least one second cross-linkablebiocompatible polymer.
 6. The method according to claim 5 wherein saidat least one second cross-linkable biocompatible polymer is a protein.7. The method according to claim 6 wherein said at least one secondcross-linkable protein is elastin.
 8. The method according to claim 1further comprising adding an active agent selected from the groupconsisting of enzyme inhibitors, anesthetic agents, medicinalneurotoxins, antioxidants, anti-infective agents, anti-inflammatoryagents, ultraviolet (UV) light blocking agents, dyes, hormones,immunosuppressants, and combinations thereof.
 9. A method of making ahydrogel for soft tissue augmentation, the method comprising: reactingat least one cross-linkable biocompatible polymer with at least onezero-length cross-linking agent at neutral pH, thereby forming across-linked biocompatible polymer comprising zero-length cross-linkedmoieties; wherein at least one said cross-linkable biocompatible polymeris hyaluronic acid (HA); and wherein the zero-length cross-linking agentis 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC).
 10. The methodaccording to claim 9, wherein the zero-length crosslinking agent isreacted with the at least one cross-linkable biocompatible polymer inthe presence of N-hydroxysuccinimide (NHS).
 11. The method according toclaim 9, wherein the neutral pH is between about 6.0 and about 8.0. 12.The method according to claim 9, wherein the neutral pH is between about6.5 and about 7.5.
 13. The method according to claim 9, wherein theneutral pH is about 7.0.
 14. The method according to claim 9, whereinthe neutral pH is maintained by a phosphate buffered saline solution.15. The method according to claim 9, further comprising at least onesecond cross-linkable biocompatible polymer.
 16. The method according toclaim 15, wherein the at least one second cross-linkable biocompatiblepolymer is a protein.
 17. The method according to claim 16, wherein theat least one second cross-linkable protein is elastin.
 18. The methodaccording to claim 9, further comprising adding an active agent selectedfrom the group consisting of enzyme inhibitors, anesthetic agents,medicinal neurotoxins, antioxidants, anti-infective agents,anti-inflammatory agents, ultraviolet (UV) light blocking agents, dyes,hormones, immunosuppressants, and combinations thereof.
 19. The methodaccording to claim 18, wherein the active agent is an anesthetic agent,which is lidocaine.